Cytomegalovirus reactivation is frequent in multiple myeloma patients treated with daratumumab‐based regimens

Abstract Background Viral reactivations are frequent in hematologial patients due to their cancer‐related and drug‐induced immunosuppressive status. Daratumumab, an anti‐CD38 monoclonal antibody, is used for multiple myeloma (MM) treatment, and causes immunosuppression by targeting CD38‐expressing normal lymphocytes. In this single‐center two‐arm real‐life experience, we evaluated incidence of cytomegalovirus (CMV) reactivation in MM patients treated with daratumumab‐based regimens as first‐ or second‐line therapy. Methods A total of 101 consecutive MM patients were included in this study and were divided into two cohorts: daratumumab and nondaratumumab‐based (control) regimens. Patients treated with >2 lines of therapies were excluded to reduce the confounding factor of multi‐treated cases. Primary endpoint was the CMV reactivation rate. Results CMV reactivation rate was significantly higher in the daratumumab cohort compared to control group (33% vs. 4%; p < 0.001), also with higher CMV‐DNA levels (>1000 UI/mL in 12% of cases; p < 0.05). However, only one subject developed a CMV disease with severe pneumonia, while 12% of patients were successfully treated with preemptive therapy with valganciclovir. No subjects in the control cohort required anti‐CMV agents (p = 0.02). Conclusion Our single‐center retrospective experience showed that daratumumab might significantly increase the risk of CMV reactivation in MM, while currently underestimated and related to morbility and mortality in MM patients under treatments. However, further validation on larger and prospective clinical trials are required.


| INTRODUCTION
Multiple myeloma (MM) accounts for 10% of all hematological malignancies, and is characterized by proliferation and accumulation in the bone marrow (BM) of clonal CD38+ CD138++ plasma cells and by overproduction of monoclonal proteins (M-protein) leading to diffuse organ damage. 1,24][5] Several mechanisms underlie this immune system deficiency, including disease-induced immunosuppression, leukopenia, impaired T-cell functions, steroid therapies, hypogammaglobulinemia, older age, severe renal dysfunction, poor performance status, and therapy-related immunosuppresion. 6,7The infectious risk is maximal during the first 3 months of therapy in all patients, decreasing in responding subjects and increasing in relapsed/refractory (RR) MM. 8 Therefore, infectious complications are a frequent cause of morbidity and mortality in MM. 9,10 Daratumumab, a fully humanized IgGκ anti-CD38 monoclonal antibody, exerts its anti-MM activity through numerous mechanisms, such as antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, direct cellular apoptosis, and modulation of extracellular ectoenzyme activity. 4CD38, a surface membrane glycoprotein, is highly expressed on malignant plasma cells, and normal B, T, and natural killer lymphocytes; therefore, its pharmacologic targeting results in neoplastic cell killing and also in immune system impairment with increased infectious risk. 11In clinical trials, the overall infectious risk under daratumumab treatment is estimated ~38% across different studies, for both newly-diagnosed and RR MM, with bacterial pneumonia as the most common infectious complication, leading to treatment discontinuation in 1%-4% of cases. 12,13Daratumumab-induced immunosuppression is also associated with an increased risk of viral reactivation, including Hepatitis B virus, Hepatitis C virus, and Herpes Zoster virus, leading to treatment delays, interruptions, or discontinuation. 125][16] After primary CMV infection, immunocompentent subjects remain silent carriers for life, unless under certain conditions, such as immunosuppression, that causes viral reactivation.As for other viral reactivations, also for CMV, the reactivation risk might be increased by daratumumab treatment. 17However, to our knowledge, few data are available on the CMV reactivation rate in MM patients, with a lack of specific prophylaxis in current guidelines, 18 and the association of viral reactivation with daratumumab therapies has not been clearly established yet.CD38 plays an important role in T cell-mediated immune responses, as it facilitates the interaction between several molecules involved in immunological synapsis and the engagement of Natural Killer cells, T and B cells. 19,20][21][22] Because of CD38 roles in physiological T cell-mediated immune responses against viral infections and reactivation, 22 we sought to investigate the incidence of CMV reactivation and disease in MM patients treated with daratumumab-based regimens as first-or second-line therapy in a single-center, two-arm, real-life experience.

| Study cohorts
A total of 101 consecutive MM patients from 2015 to November 2023 under active first-or second-line treatment were included in this single-center retrospective study, and were divided into two cohorts: daratumumab and nondaratumumab-based (control group) regimens.Patients treated with >2 therapy lines were excluded to reduce confounding factors, such as advanced disease, very poor patient conditions, and deep immunosuppressive status. 23A control group was employed to investigate the impact of other factors involved in viral reactivation, including high-dose glucocorticoids or the use of proteasome inhibitors. 24,25Inclusion criteria were: age ≥ 18 years old; diagnosis of MM according to the International Myeloma Working Group (IMWG) criteria 26 ; active first-or second-line anti-MM therapy outside clinical trials.This study was conducted in accordance with the Declaration of Helsinki, and protocols approved by our Ethics Committee "Campania Sud" (Brusciano, Naples, Italy; prot./SCCEn. 24,988).All patients provided written informed consent.

| CMV quantification and serology
Lymphocyte count and anti-CMV immunoglobulin G (IgG) levels were measured in patients at diagnosis and at reactivation.Hypogammaglobulinemia was defined as total IgG levels <400 mg/dL.Plasma CMV-DNA copy number was quantified by real-time TaqMan CMV-DNA PCR according to manufacturers' instructions (Roche), monthly at every chemotherapy cycle, as per our institution's guidelines.After diagnosis of CMV reactivation, CMV-DNA levels were assessed weekly until negativization.The instrument cut-off for positive results was CMV-DNA copy number > 137 copies/μL, as previously described. 16CMV reactivation was defined as CMV-DNA copy number > 137 copies/μL in anti-CMV IgG+ patients without organ damage, while CMV disease was defined when CMV-related organ damage occurred, including pneumonia, gastrointestinal disease, hepatitis, retinitis, central nervous system disease, nephritis, myocarditis, and pancreatitis, associated to the detection of CMV-DNA on the involved organ. 27Anti-CMV agents were initiated when weekly progressively increasing in CMV-DNA levels were observed.

| Endpoints and statistical analysis
The primary endpoint was incidence of CMV reactivation with or without CMV-related organ damage, and secondary endpoint was the incidence of CMV disease.Data were collected in spreadsheets and were analyzed using R statistical software (v.4.0.5;RStudio) and SPSS (v.25; IBM).Continuous variables were expressed as mean or median and compared with the Wilcoxon rank-sum or student's t-test.Categorical variables were expressed as counts and percentages and compared using Chi-square or Fisher's exact test as appropriate.A p value of <0.05 was considered statistically significant.
Antiviral prophylaxis with acyclovir was less frequently employed in the daratumumab arm (62% vs. 86%; p = 0.02), while CMV reactivation rate was significantly higher in this cohort (33% vs. 4%; p = 0.001), with six patients (12%) showing a CMV-DNA copy number > 1000 UI/mL (12% vs. 0%, daratumumab vs. control group; p = 0.03).Time to CMV reactivation was shorter in the daratumumab cohort, although this was not significant (29 vs. 54 days, daratumumab versus control group; p = 0.64).Moreover, the only subject who developed CMV disease was in the daratumumab arm, and the patient had severe grade III interstitial pneumonia associated with high CMV-DNA levels (141.000UI/mL) treated with intravenous ganciclovir.Among the remaining patients with CMV reactivation (6 out of 7), preemptive therapy with oral valganciclovir was successfully employed.T A B L E 1 Patients' characteristics at enrollment.

| DISCUSSION
9][30][31][32] Although the risk of bacterial infections and hepatitis virus or HZV reactivation has been clearly assessed, few data are available on CMV reactivation risk, without a clear recommendation for anti-CMV prophylaxis. 13,17In this single-center, two-arm, retrospective, real-life study, we investigated the CMV reactivation rate in MM patients treated with the anti-CD38 monoclonal antibody daratumumab as first-or second-line therapy, and outcomes were compared to a daratumumab-naïve control group.
To the best of our knowledge, our real-life experience is the first study investigating CMV reactivation risk by comparing the results of daratumumab-treated to daratumumab-naïve MM patients in a fairly large number of nonmultirefractory subjects (<2 therapy lines; N = 51 and N = 50, respectively).Multi-treated patients were excluded, as their very poor clinical conditions, uncontrolled disease, or several previous lines of immunosuppressive therapy might have been confounding factors, while other risk factors for immunosuppression, such as diabetes or dialysis, were balanced between groups.
CMV reactivation in daratumumab-treated MM patients has only been anectodically reported with only single case in a heavily pretreated MM patient under single-agent daratumumab therapy (7th line) and treated with intravenous ganciclovir 5 mg/kg twice a day, and with other three heavily pretreated MM cases of severe CMV-related enterocolitis requiring prolonged anti-CMV treatment. 33,34In other previous studies, in a cohort of 13 RR MM patients (median of previous therapy lines, 5) under daratumumab-containing regimens, the CMV reactivation rate has been assessed to 38%, while in a cohort of 15 MM patients with 3.5 median prior therapy lines, the reactivation rate has been reported at 73%, a very high incidence likely because 47% of these subjects have been previously received autologous stem cell transplantation. 18,35,36Conversely, in our study, rates of prior autologous stem cell transplantation were very low in both cohorts, without significant differences between groups.CMV reactivation/infection might also frequently cooccur with Epstein-barr virus reactivation in MM patients under daratumumab treatment (17% of cases), especially in multi-treated RR MM. 37 Proteasome inhibitors can also increase the risk of CMV reactivation in plasma cell dyscrasias (MM or light chain amyloidosis) 25 ; however, in our control group, almost the entire cohort received proteasome inhibitors (bortezomib, 84%; and carfilzomib, 16%) without influencing CMV reactivation risk, as the rate was much lower (4%) than that previously reported for proteasome inhibitor-treated MM patients (39%). 25aratumumab is given as 1 weekly infusion during cycles 1 to 2 (weeks 1 to 8), then as 1 infusion every 2 weeks (twice per 4-week cycle; cycles 3 to 6; weeks 9 to 24), and subsequently as 1 infusion every 4 weeks (cycle 7+; week 25+ until disease progression), according to approved dosing schedule.In our daratumumab group, median time to CMV reactivation was 29 days, that was during weekly daratumumab administration, suggesting that closer dosing causes a more pronounced immunosuppression and increases the risk of viral reactivation, because of the important role of CD38 in T cell-mediated immune responses. 22n our report, only one patient suffered CMV-related pneumonia under dara-VTD regimen; detection of CMV-DNA in bronchoalveolar lavage was not performed because of suggestive radiological signs of interstitial pneumonia by lung CT scan, and a very high plasma viral load (141.000UI/mL).Our patient was successfully treated with intravenous ganciclovir, and restarted anti-MM treatment after pneumonia resolution.In all other cases with an increased viral load, preemptive therapy was promptly started to reduce the risk of CMV disease development.
Our study has several limitations: (i) retrospective nature of this investigation, although clinical characteristics were well-balanced between groups; (ii) unavailability of anti-CMV IgG serostatus at baseline for the entire study population, thus we can not exclude primary CMV infection rather than reactivations, although primary disease occurs in younger subjects, as over half of general adult population has been infected by age 40 38 ; (iii) preemptive therapy was started based on clinical decision, considering various clinical and laboratory factors (e.g., lymphocyte count), 39 and not on a specific CMV-DNA copy number level; and (iv) acyclovir prophylaxis was more frequently performed in control group, even though all patients received low acyclovir doses (400 mg/twice daily or lower doses, according to renal function).
In conclusions, daratumumab can increase the risk of viral infections and reactivations, including CMV reactivation.In our retrospective single-center two-arm observational study, a marked increase in CMV reactivation was reported in daratumumab-treated MM patients, that could be successfully reduced with a prompt initiation of preemptive therapy by preventing CMV-related disease development.Indeed, CMV reactivation might silently occur and lead to cytopenias or interstitial pneumonias, thus a regular CMV-DNA monitoring could both reduce antiviral prophylaxis-related toxicity as therapy would be started as needed, and CMV disease development rates.
MM patients treated with anti-CD38 agents are at higher risk of infectious complications, especially pneumonia not Varicella-Zoster Virus-related, without an associated higher mortality rate. 12Other single-center real-life experiences or case series have reported that CMV reactivation can be frequent in anti-CD38 agent treated MM subjects in up to 38% of cases 35,36,40,41 ; however, symptoms are commonly mild with fever, while CMV disease development is rare. 40Despite severe disease is unfrequently observed and CMV monitoring could be expensive, time consuming, and not easily applicable to routinely clinical practice, it has been shown that CMV reactivation negatively impact on clinical outcomes of MM patients, as dose reduction or drug discontinuation might be required until disease resolution. 40A close monitoring might early identify mild CMV-related symptoms (e.g., cytopenias or diarrhea), that could be gone unrecognized or misattributed as drug-induced toxicity, and could allow prompt initiation of anti-viral agents before severe disease development.Moreover, CMV reactivation might be more frequent than already reported, because CMV monitoring is not commonly employed in clinical practice, while when performed, viral reactivation can be found in a high percentage of treated subjects, as reported from ongoing clinical trials with daratumumab in association with bispecific antibodies. 42,43Therefore, CMV-DNA level assessment before starting each chemotherapy cycle could be a valuable laboratory tool for monitoring viral reactivation risk in MM patients under daratumumab treatment.However, further validation on larger and prospective clinical trials are required.
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